Background: The increase in carbapenemase producing Enterobacteriaceae and Pseudomonas aeruginosa is a significant threat to modern medicine. A rapid detection of carbapenemase production in Klebsiella pneumoniae and Pseudomonas aeruginosa is of importance for the institution of correct antibiotic treatment and infection control measures. Results: Standardised inoculums of K. pneumoniae or P. aeruginosa were incubated at 37°C with ertapenem in 15 and 120 min followed by centrifugation. The supernatant was applied on a steel target plate, covered with HCCA matrix and analysed using a MicroflexTM (Bruker Daltonics) in the mass range of 4–600 Da. The assay detected and separated KPC from other carbapenemases in K. pneumoniae after only 15 min incubation. In P. aeruginosa, however, only 8/14 isolates of VIM-producing P. aeruginosa were detected. None of the tested carbapenemase negative isolates displayed a pattern of hydrolysis of ertapenem. Conclusions: This assay allows for a very rapid detection and verification of KPC (45 min including the preparation steps) and MBL production (150 min) in K. pneumoniae and can be performed using standard matrix. However, the study revealed the need for optimization of the substrate/species combination in assays for the detection of carbapenemases in P. aeruginosa using MALDI-TOF.